Agrobacterium-mediated gene introduction is a method for transforming plants through the use of a function of Agrobacterium. A soil bacterium Agrobacterium (Agrobacterium tumefaciens) functions in such a manner that the T-DNA forming a part of its Ti (tumor-inducing) plasmid involved in the pathogenicity of the Agrobacterium is integrated into the genome of a plant when it infects the plant. Agrobacterium-mediated plant transformation is a method for introducing a desired gene into the genome of a plant through the use of the function of Agrobacterium described above by constructing a transforming plasmid in which the T-DNA region of the Ti plasmid is replaced by the gene desired to be introduced into the plant genome and then using Agrobacterium prepared to carry the transforming plasmid in place of the Ti plasmid.
Agrobacterium-mediated plant transformation was originally developed mainly as a method for transforming dicotyledons because Agrobacterium were thought to infect only dicotyledons but not monocotyledons. Subsequently, various attempts for Agrobacterium-mediated gene introduction into monocotyledons were also made, and super-binary vectors having a part of the virulent genes of super-virulent Agrobacterium strains were developed and reported to be useful for stably transforming even monocotyledons such as rice and maize with relatively high efficiency (e.g., see Japanese Patent No. 2,649,287; Japanese Patent No. 3,329,819; Hiei, Y., et al., (1994), The Plant Journal, Vol. 6, p. 271-282; and Ishida, Y., et al., (1996), Nature Biotechnology, Vol. 4, p. 745-750). Successful examples of Agrobacterium-mediated transformation of monocotyledons such as wheat, barley and sorghum were also reported (e.g., see Cheng, M., et al., (1997), Plant Physiol., Vol. 115, p. 971-980; Tingay, S., et al., (1997), Plant J., Vol. 11, p. 1369-1376; and Zhao, Z-Y., et al., (2000), Plant Mol. Biol., Vol. 44, p. 789-798), and Agrobacterium-mediated transformation also began to be widely applied to monocotyledons.
Agrobacterium-mediated transformation generally has many advantageous features such as high efficiency, low copy number transgenes, transducibility of such a specific region as T-DNA without being fragmented, and short-term culture for obtaining transformants resulting in less mutation during culture. Thus, it is now widely used as the most useful means for transforming many plant species irrespective of whether they are dicotyledonous or monocotyledonous.
Agrobacterium-mediated transformation is equally performed in all plants by contacting a material tissue with an Agrobacterium suspension, coculturing them and then selecting a transformed cell to produce a transformed plant, though the materials and the compositions of the culture media vary with plant species. Generally, the plant tissue used as a material is infected with Agrobacterium without any special treatment except for optional sterilization (e.g., see Rogers, S. G., et al., (1988), Method for Plant Molecular Biology, p. 423-436, CA: Academic Press Inc.; Visser, R. G. F., (1991), Plant Tissue Culture Manual, B5:1-9, Kluwer Academic Publishers; McCormick, S., (1991), Plant Tissue Culture Manual, B6:1-9, Kluwer Academic Publishers; and Lindsey, K., et al., (1991), Plant Tissue Culture Manual, B7:1-13, Kluwer Academic Publishers).
Agrobacterium-mediated transformation has been reported for many plant species, but has the disadvantage that the transformation efficiency widely varies with plant species, genotypes and material tissues (e.g., see Potrykus, I., et al., (1998), Agricultural Biotechnology, NY: Mercel Dekker Inc., p. 119-159). It is important to develop a technology enabling transformed plants to be stably obtained with high efficiency throughout the year because many transformed plants must be produced when a cultivar containing a practical gene is to be bred. Moreover, transformation methods independent of plant species and genotypes would be very useful for efficiently breeding practical cultivars. Development of transformation methods independent of material plant tissues would also be required for efficient transformation.
Thus, it is important to develop a method capable of improving gene transduction efficiency or transforming even plant species or genotypes involving difficulty in gene transduction. Many techniques for efficiently obtaining transformed plants have already been reported in various aspects such as the adaptation of the compositions of culture media, the alteration of marker genes, or promoters or the investigation of materials and treatment methods for materials. For example, treatment methods for materials by injuring tissues to improve infection efficiency or by centrifuging (e.g., see International Publication No. WO02/12520; Japanese Patent Public Disclosure No. 2000-342256) or heating (e.g., see Japanese Patent Public Disclosure No. 2000-342255; Japanese Patent Public Disclosure No. 2000-342253) plant tissues without injuring them have been reported. The present inventors previously found that pressurization of plant tissues is useful for improving gene transduction efficiency (with the results unpublished).
Among monocotyledons, maize had the disadvantage that the Agrobacterium-mediated transformation efficiency is lower than that of rice. Various attempts have already been made to improve the Agrobacterium-mediated transformation efficiency of maize (e.g., see Negrotto, D., et al., (2000), Plant Cell Reports, Vol. 19, p. 798-803; Zhao, Z-Y., et al., (2001), Mol. Breed., Vol. 8, p. 323-333; Frame, B. R., et al., (2002), Plant Physiol., Vol. 129, p. 13-22; and , Ishida, Y., et al., (2003), Plant Biotechnology, Vol. 14, p. 57-66). Various previous attempts to improve the Agrobacterium-mediated transformation efficiency of maize include selecting transformed cells on N6 basal medium (e.g., see Zhao, Z-Y., et al., (2001), Mol. Breed., Vol. 8, p. 323-333), adding silver nitrate and carbenicillin to culture media (e.g., see Zhao, Z-Y., et al., (2001), Mol. Breed., Vol. 8, p. 323-333; and , Ishida, Y., et al., (2003), Plant Biotechnology, Vol. 14, p. 57-66), adding cysteine to coculture media (e.g., see Frame, B. R., et al., (2002), Plant Physiol., Vol. 129, p. 13-22), etc., but the resulting effects are still low. Transformation methods with higher transformation efficiency would be desirable especially for major crops associated with low transformation efficiency such as maize not only when practical transformed plants are to be produced but also when the effect of a novel gene is to be tested.
Copper sulfate is contained as a minor salt in a wide variety of media. Normally, the concentration of copper sulfate in plant tissue culture media is 0.1 μM. Recent report shows that various effects were observed when adding copper sulfate at 50-fold to 500-fold higher concentrations than normal levels to media in tissue cultures and transformation tests of monocotyledons. Ghaemi et al. (see Ghaemi, M., et al., (1994), Plant Cell, Tissue and Organ Culture, Vol. 36, p. 355-359) reported that embryoid formation increases by culturing anthers of wheat in a medium containing 10 mg/l copper sulfate and 2.5-5 mg/l silver nitrate. Zhang et al. (see Zhang, S., et al., (1999), Plant Cell Reports, Vol. 18, p. 959-966) reported that the induction ratio of shoot meristematic cultures (SMCs) increases by culturing shoots having emerged from ripe seeds of barley in a medium containing 5 μM copper sulfate and 30 g/l maltose. They describe that maltose is effective for decreasing brown tissue and that copper sulfate is effective for promoting shoot growth when shoot meristematic cultures are induced. It was also reported that the regeneration ratio or the number of regenerated plants per callus increases in calli obtained by culturing immature embryos of barley (e.g., see Dahleen, L. S., (1995), Plant Cell, Tissue and Organ Culture, Vol. 43, p. 267-269; and, Cho, M-J., et al., (1998), Plant Science, Vol. 138, p. 229-244) and rice (e.g., see Sahrawat, A. K. and Chand, S., (1999), J. Plant Physiol., Vol. 154, p. 517-522) in media containing copper sulfate. It was also reported that green tissues having regeneration potential induced in media containing copper sulfate are suitable as materials for transformation (e.g., see Visser, R. G. F., (1991), Plant Tissue Culture Manual, B5:1-9, Kluwer Academic Publishers; and, McCormick, S., (1991), Plant Tissue Culture Manual, B6:1-9, Kluwer Academic Publishers).
Ishida et al. (see Ishida, Y., et al., (2003), Plant Biotechnology, Vol. 20, p. 57-66) investigated callus formation from immature embryos by culturing immature embryos of maize (cultivar: H99) inoculated and co-cultured with Agrobacterium, on media containing 1-100 μM copper sulfate. The callus formation improved in media containing 1-10 μM copper sulfate, but slightly.
As described above, previous reports showed that various effects were observed in tissue cultures of monocotyledons by adding high concentrations of copper sulfate to media. However, there has been no report on the effects of adding a metal salt containing copper ion on gene introduction efficiency and/or transformation efficiency.